利用CRISPR/Cas9系统在小鼠胚胎干细胞中进行miR-22的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-0964

摘要/Abstract

摘要： 旨在构造neomycin筛选标记的sgRNA表达载体,并利用CRISPR/Cas9技术构建miR-22缺失的小鼠胚胎干细胞,探究miR-22在小鼠胚胎干细胞中的调控作用。首先通过点突变、搭桥PCR等手段,构造neomycin筛选标记的sgRNA表达载体,并获得靶向敲除miR-22的sgRNA表达载体,电转至稳转Cas9的小鼠胚胎干细胞中;其次经药物筛选、基因型鉴定等步骤筛选miR-22纯合缺失的小鼠胚胎干细胞。RT-qPCR手段证实miR-22在小鼠胚胎干细胞中被成功敲除,纯合突变体的比例约为6.67%。此外,miR-22缺失并未影响胚胎干细胞的细胞形态以及Oct4、Sox2和Nanog等干性基因的表达。因此,miR-22对小鼠胚胎干细胞的干性维持并非必需,而对胚胎干细胞谱系分化和命运决定的影响还有待进一步研究。

关键词: 小鼠胚胎干细胞, CRISPR/Cas9, miR-22

Abstract: This study aims to construct neomycin-resistant sgRNA expression plasmids and to generate miR-22 knockout mouse embryonic stem cells（mESCs）with CRISPR/Cas9 for examining miR-22’s regulatory roles. First,point mutation and overlap PCR were performed to construct neomycin-resistant sgRNA expression plasmids. Then,sgRNA expression plasmids of miR-22 knockout were electroporated into mESCs with stable Cas9 expression. After neomycin selection and PCR genotyping,the miR-22 homozygous knockout mESCs were screened. The homozygous deletion was confirmed by RT-qPCR experiments and the efficiency of homozygous knockout was about 6.67%. In addition,the deletion of miR-22 presented no obvious effect on the morphology of mESC as well as the expressions of pluripotent markers including Oct4,Sox2 and Nanog. Together,miR-22 may be not necessarily required for the maintenance of embryonic stem cell,while the roles of miR-22 in controlling mESC differentiation and cell fate decision need further identification.

Key words: mouse embryonic stem cell, CRISPR/Cas9, miR-22

张雪,陈亮亮,戴红霞,张文胜,任文燕. 利用CRISPR/Cas9系统在小鼠胚胎干细胞中进行miR-22的功能研究[J]. 生物技术通报, 2018, 34(5): 71-79.

ZHANG Xue, CHEN Liang-liang, DAI Hong-xia, ZHANG Wen-sheng, REN Wen-yan. Functional Study of miR-22 in Mouse Embryonic Stem Cell with CRISPR/Cas9 System[J]. Biotechnology Bulletin, 2018, 34(5): 71-79.

参考文献

[1] Martin GR.Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc Natl Acad Sci USA, 1981, 78(12):7634-7638.
[2] Martello G, Smith A.The nature of embryonic stem cells[J]. Annu Rev Cell Dev Biol, 2014, 30:647-75.
[3] Hackett JA, Surani MA.Regulatory principles of pluripotency:from the ground state up[J]. Cell Stem Cell, 2014, 15(4):416-430.
[4] Huang G, Ye S, Zhou X, et al.Molecular basis of embryonic stem cell self-renewal:from signaling pathways to pluripotency network[J]. Cell Mol Life Sci, 2015, 72(9):1741-1757.
[5] Chen T, Dent SY.Chromatin modifiers and remodellers:regulators of cellular differentiation[J]. Nat Rev Genet, 2014, 15(2):93-106.
[6] Gurtan AM, Sharp PA.The role of miRNAs in regulating gene expression networks[J]. J Mol Biol, 2013, 425(19):3582-600.
[7] Ling B, Wang GX, Long G, et al.Tumor suppressor miR-22 suppresses lung cancer cell progression through post-transcriptional regulation of ErbB3[J]. J Cancer Res Clin Oncol, 2012, 138(8):1355-1361.
[8] Tang Y, Liu X, Su B, et al.microRNA-22 acts as a metastasis suppressor by targeting metadherin in gastric cancer[J]. Mol Med Rep, 2015, 11(1):454-460.
[9] Xin M, Qiao Z, Li J, et al.miR-22 inhibits tumor growth and metastasis by targeting ATP citrate lyase:evidence in osteosarcoma, prostate cancer, cervical cancer and lung cancer[J]. Oncotarget, 2016, 7(28):44252-44265.
[10] Guo S, Bai R, Liu W, et al.miR-22 inhibits osteosarcoma cell proliferation and migration by targeting HMGB1 and inhibiting HMGB1-mediated autophagy[J]. Tumour Biol, 2014, 35(7):7025-7034.
[11] Li G, Wang G, Ma L, et al.miR-22 regulates starvation-induced autophagy and apoptosis in cardiomyocytes by targeting p38alpha[J]. Biochem Biophys Res Commun, 2016, 478(3):1165-1172.
[12] Ji D, Li B, Shao Q, et al.MiR-22 Suppresses BMP7 in the Development of Cirrhosis[J]. Cell Physiol Biochem, 2015, 36(3):1026-1036.
[13] Zhao H, Wen G, Huang Y, et al.MicroRNA-22 regulates smooth muscle cell differentiation from stem cells by targeting methyl CpG-binding protein 2[J]. Arterioscler Thromb Vasc Biol, 2015, 35
(4):918-929.
[14] Horvath P, Barrangou R.CRISPR/Cas, the immune system of bacteria and archaea[J]. Science, 2010, 327(5962):167-170.
[15] Marraffini LA, Sontheimer EJ.CRISPR interference:RNA-directed adaptive immunity in bacteria and archaea[J]. Nat Rev Genet, 2010, 11(3):181-190.
[16] Mei Y, Wang Y, Chen H, et al.Recent Progress in CRISPR/Cas9 Technology[J]. J Genet Genomics, 2016, 43(2):63-75.
[17] Qi LS, Larson MH, Gilbert LA, et al.Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression[J]. Cell, 2013, 152(5):1173-1183.
[18] Ran FA, Hsu PD, Lin CY, et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity[J]. Cell, 2013, 154(6):1380-1389.
[19] Wei C, Liu J, Yu Z, et al.TALEN or Cas9 - rapid, efficient and specific choices for genome modifications[J]. J Genet Genomics, 2013, 40(6):281-289.
[20] Cai M, Yang Y.Targeted genome editing tools for disease modeling and gene therapy[J]. Curr Gene Ther, 2014, 14(1):2-9.
[21] Doudna JA, Charpentier E.Genome editing. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346(6213):1258096.
[22] Wang T, Wei JJ, Sabatini DM, et al.Genetic screens in human cells using the CRISPR-Cas9 system[J]. Science, 2014, 343(6166):80-84.
[23] Xiao A, Zhang B.Generation of targeted genomic deletions through CRISPR/Cas system in Zebrafish[J]. Methods Mol Biol, 2016, 1451:65-79.
[24] Bassett AR, Tibbit C, Ponting CP, et al.Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[J]. Cell Rep, 2014, 6(6):1178-1179.
[25] Wang H, Yang H, Shivalila CS, et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering[J]. Cell, 2013, 153(4):910-918.
[26] Ryder P, McHale M, Fort A, et al. Generation of stable nulliplex autopolyploid lines of Arabidopsis thaliana using CRISPR/Cas9 genome editing[J]. Plant Cell Rep, 2017, 36(6):1005-1008.
[27] Wei W, Xin H, Roy B, et al.Heritable genome editing with CRISPR/Cas9 in the silkworm, Bombyx mori[J]. PLoS One, 2014, 9(7):e101210.
[28] Gilles AF, Schinko JB, Averof M.Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum[J]. Development, 2015, 142(16):2832-2839.
[29] Boyer LA, Lee TI, Cole MF, et al.Core transcriptional regulatory circuitry in human embryonic stem cells[J]. Cell, 2005, 122(6):947-956.
[30] Gangaraju VK, Lin H.MicroRNAs:key regulators of stem cells[J]. Nat Rev Mol Cell Biol, 2009, 10(2):116-125.
[31] Stadler B, Ivanovska I, Mehta K, et al.Characterization of microRNAs involved in embryonic stem cell states[J]. Stem Cells Dev, 2010, 19(7):935-950.
[32] Houbaviy HB, Murray MF, Sharp PA.Embryonic stem cell-specific MicroRNAs[J]. Dev Cell, 2003, 5(2):351-358.
[33] Song SJ, Ito K, Ala U, et al.The oncogenic microRNA miR-22 targets the TET2 tumor suppressor to promote hematopoietic stem cell self-renewal and transformation[J]. Cell Stem Cell, 2013, 13(1):87-101.
[34] Huang S, Wang S, Bian C, et al.Upregulation of miR-22 promotes osteogenic differentiation and inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells by repressing HDAC6 protein expression[J]. Stem Cells Dev, 2012, 21(13):2531-40.